Genetic and Phylogenetic Characterization of the M Gene of Influenza A Virus Isolated from Iranian Patients.

Background: A few studies have been done on the molecular analysis of Iranian influenza A isolates M gene. Methods: In 2014, nasal swabs collected from outpatients with clinical symptoms in the hospital clinics of Tehran, Iran were subjected for influenza detection and subtyping using Real-Time RT-PCR. Sequence and phylogenetic analysis performed on four randomly selected isolates from each subtype (H1N1 and H3N2) using neighbor-joining method. Results: Phylogenetic dendrograms drawn based on M nucleotide sequence of H1N1 isolates showed close relatedness with Omanian isolates while the most isolates of H3N2 have clustered with Kuwait isolates and isolates from outside of geographical location. Amino acid sequence analysis showed S31N substitution in all isolates rendering the virus resistant to adamantanes. Conclusion: This study determined the sequence identity and phylogenetic relatedness of M gene sequence got from Iranian influenza A isolates to elucidate the modality of relationship of this gene in comparison with its counterparts from other regions.


Introduction
Currently, two Influenza A virus subtypes, H1N1 and H3N2, are circulating among humans. Influenza A viruses are known as a major cause of acute respiratory disease worldwide. Around 1 billion cases of seasonal influenza infection occur each year, with around 3-5 million cases of severe illness, and 300000-500000 deaths (1,2). Antiviral drugs are used to reduce the duration of disease, viral shedding, rate of hospitalization and death in Influenza A virus-infected persons (3). These drugs are also used for prevention of influenza virus infection (4). The seventh RNA segment of influenza A virus code two M proteins. While M1 is matrix protein and plays an essential role in virion assembly, budding and release, M2 is ion channel in the envelope and plays important role in virus replication cycle. M2 is activated by low pH of the endosome and conduct protons across the envelope, which results in the acidification of the viral interior. This acidification weakens electrostatic interaction between M1 and ribonucleoprotein (RNP) complexes and releases RNP into cytosol. M2 of influenza A virus is a 97-residue protein and composed of extracellular N-terminal (residue 1-23), transmembrane (residue 24-46), and intracellular C-terminal (residue 47-97) segments (5). Some of amino acid substitutions in the transmembrane segment (position 26, 27, 30, 31, 34, 38 or 41) were shown in amantadine-resistant strains of influenza A virus (4). Adamantanes, amantadine and rimantadine, are M2 protein inhibitors known for many decades to inhibit most influenza A viruses (3,6). Unfortunately, resistance to amantadine and to its 10-fold more active derivative, rimantadine, develops with increased use in humans and animals. Nowadays, several new kinds of antiviral compounds are being designed and developed against influenza viruses, which block the other critical steps of the viral life cycle (2,6). Several articles have been published regarding phylogenetic and molecular analysis of the influenza A virus M gene, isolated from different countries. However, there are a few studies from Iran that focused on M2 sequences (7,8). Here, we performed a study on the molecular and phylogenetic characterization of the whole M fragment including M2 gene of human influenza A viruses (H1N1 and H3N2) isolated from Iranian patients.

Isolation of Influenza viruses
Influenza viruses were isolated by standard methods from nasal swabs of outpatients with clinical symptoms in the hospital clinics of Tehran, Iran, during fall and winter 2014. Briefly, the nasal swab samples were tested for presence of Influenza A, H1N1 and H3N2 subtypes using Real-Time Reverse Transcription PCR (RT-PCR) according to instructions recommended by WHO (9). Virus from positive samples was propagated and isolated in MDCK cells. Hemagglutination assay (HA) was performed using a 0.5% suspension of the chicken erythrocytes to confirm virus growth. Positive cultures were harvested and stored at -70°C until further analysis. Four H1N1 and four H3N2 positive samples were randomly selected and subjected for next stages.

Amplification of M gene
Viral RNA was extracted from 300 µl of the supernatant of cell cultures for each sample using a commercial viral nucleic acid extraction kit (YTA, Iran) and eluted in 20 µl DEPC treated water. Specific primers for amplification of complete M fragment of both genotypes of human influenza viruses, H1N1 and H3N2, were designed using the BioEdit software (version 7.2.5). The list of primers and their characteristics are shown in Table 1. The one-step RT-PCR was done using YTA master mix according to the manufacturer's recommendation, under the following profile: reverse transcription at 50 ºC for 30 min, reverse transcriptase termination at 95 ºC for 15 min and HotStart Taq amplification in 40 cycles (95 ºC for 20 sec, 58 ºC for 40 sec, and 72 °C for 1 min) concluded by a final extension step at 72 °C for 10 min. PCR products were analyzed by gel electrophoresis on a 1.5% agarose gel and then purified by a GF-1 PCR Clean-up Kit (Vivantis, Malaysia) according to the manufacturer's instruction prior to sequencing.

Phylogenetic analysis
Phylogenetic trees of M genes were constructed using the neighbor-joining method within MEGA 5.0 using Kimura two-parameter method. The stability of nodes and tree branching was determined by bootstrapping using 1000 repeats of drawing.
In addition, the amino acid sequences of the matrix proteins were compared, particularly in terms of differences in M2 (Table 2) which distinguish resistant variants of H1N1 and H3N2 viruses circulating during that period.

Discussion
The matrix gene encodes for matrix (M1) and ion channel (M2) proteins both with multiple crucial functions. Since M1 protein plays an essential role in virus assembly and budding, any change in M1 can have deleterious effect on virus survival so its gene is conserved with low evolutionary rate into host-specific lineages (10,11 (7). The present study showed that all strains of H1N1 and H3N2 subtypes contained the amino acid substitutions S31N, as expected. However, other mutations in the transmembrane region of M2 linked with adamantane resistance have not been seen in all studied H1N1 and H3N2 viruses (7,17,18). Adamantanes-resistance markers are combined with sequence data of HA and M genes so it may be required to elucidate the genetic association among HA and M2 genes and/or other genes in the generation and spread of communitycirculating such resistant strains. Due to the dramatic increase in resistant isolates, adamantanes not be used for the treatment of influenza, except in selected circumstances (19). However, providing the M fragment sequence data of viruses isolated from different geographic regions and defining their mutation profile would be very helpful to realize the emerging, spread and evolutionary scenario of human influenza A viruses.

Conclusion
This study determined the sequence identity and phylogenetic relatedness of M gene got from influenza A circulating trough flu outbreak 2014 in Iran to elucidate the modality of relationship of this gene in comparison with its counterparts from other geographic regions.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or fal-sification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.